1. Field of the Invention
The present invention relates to compositions and methods for treating mammalian disease conditions that are debilitating, fatal, hereditary, degenerative and/or undesirable. More specifically, the present invention relates to the transplantation of normal, or genetically transduced, or cytocline-converted myogenic cells into malfunctioning, and/or degenerative tissues or organs.
2. Description of the Prior Art
MYOBLAST PROPERTIES
In mammals, myoblasts are the only cell type which divide extensively, migrate, fuse naturally to form syncytia, lose their major histocompatibility Class I (MHC 1) antigens soon after fusion, and develop to occupy 50% of the body weight in humans. These combined properties render myoblasts ideal for gene transfer and somatic cell therapy (SCT). Myoblast therapy is a combined SCT and gene therapy.
MYOBLAST THERAPY
Although the role of myoblasts/satellite cells in myogenesis and muscle regeneration dated back to the early 1960s (Konigsberg, I. R., Science, 140:1273 (1963). Mauro, A. J., Biophys. Biochem. Cytol., 9:493-495 (1961)), their use in animal therapy was not reported until 1978 (Law, P. K., Exp. Neurol., 60:231-243 1978)).
The first myoblast transfer therapy (MTT) on a Duchenne muscular dystrophy (DMD) boy on Feb. 15, 1990 marked the first clinical trial on human gene transfer. Its success was reported (Law, P. K. et al., Lancet, 336:114-115 (1990); Kolata, G. The New York Times, Sunday, (Jun. 3, 1990)). Unlike bone marrow transplant which strictly replaces genetically abnormal cells with normal ones, MTT actually inserts, through natural cell fusion, all the normal genes within the nuclei of the donor myoblasts into the dystrophic myofibers to repair them. In addition, donor myoblasts also fuse among themselves, forming genetically normal myofibers to replenish degenerated ones. Thus, full complements of normal genes are integrated, through a natural developmental process of regeneration, into the abnormal cells and into the abnormal organ.
The U.S. Patent Office issued to this inventor a patent (U.S. Pat. No. 5,130,141) entitled "Composition for and methods of treating muscle degeneration and weakness" on Jul. 14, 1992.
In October, 1993, the Food and Drug Administration (FDA) officially began regulating somatic cell therapy (SCT) with a definition of "autologous, allogenic, or xenogeneic cells that have been propagated, expanded, selected, pharmacologically treated, or otherwise altered in biological characteristics ex vivo to be administered to humans and applicable to the prevention, treatment, cure, diagnosis, or mitigation of disease or injuries." (Federal Register, 58:53248-53251 (1993)).
MTT falls under the umbrella of SCT and myoblasts and its physical, genetic or chemical derivatives become potential biologics in the treatment of mammalian diseases.
As of May 25, 1994 the FDA has granted permission for Cell Therapy Research Foundation (CTRF) to charge $63,806 per subject. CTRF is an non-profit 501 (c) (3) research foundation founded by the inventor in 1991. Authorization by the FDA for CTRF to recover costs from subjects of these clinical trials is extremely important to establish the scientific credibility MTT and CTRF deserve, quoting the Jun. 17, 1994 edition of the Memphis Health Care News, "Permission to bill for an Investigational product is granted rarely," says FDA spokesman Monica Revelle, "Applicants must endure numerous procedures, and must have what looks like a viable product at the end of the rainbow. It's used mainly to support testing of promising technology by small companies." This statement was made in regard to research at CTRF.
At this time CTRF holds the first and only FDA-approved human clinical trial under an Investigational New Drug (IND) application on MTT. It is extremely important to realize that CTRF has been working closely with the FDA to establish criteria and policies in the approval process of this IND for genetic cell therapy. The use of viral vector mediated gene therapy on human neuromuscular diseases has not met FDA approval.
CELL THERAPY WITH MYOBLASTS
The cell is the basic unit of all lives. It is that infinitely small entity which life is made of. With the immense wisdom and knowledge of the human race, we have not been able to produce a living cell from nonliving ingredients such as DNA, ions, and biochemicals.
Cell Culture is the only method known to man for the replication of cells in vitro. With proper techniques and precautions, normal or transformed cells can be cultured in sufficient quantity to repair, and to replenish degenerates and wounds.
Cell transplantation bridges the gap between in vitro and in vivo systems, and allows propagation of "new life" in degenerative tissues or organs of the living yet genetically defective or injured body.
Cell fusion transfers all the normal genes within the nucleus like delivering a repair kit to the abnormal cell. It is important to recognize that, for proper installation and future operation, the software packaged in the chromosomes needs other cell organelles as hardware to operate.
Correction of a gene defect occurs spontaneously at the cellular level after cell fusion. The natural integration, regulation and expression of the full complement of over 80,000 normal genes impart the normal phenotypes onto the heterokaryon. Protein(s) or factor(s) that were not produced by the host genome because of the genetic defect are now produced by the donor genome that is normal. Various cofactors derived from expression of the other genes corroborate to restore the normal phenotype.
GENE THERAPY WITH MYOBLASTS
The use of myoblasts as gene transfer vehicles has been researched by this inventor extensively. In mammals, myoblasts are the only cell type which divide extensively, migrate, fuse naturally to form syncytia, lose MHC-1 antigens soon after fusion, and develop to occupy 50% of the body weight in humans. These combined properties render myoblasts ideal for gene transfer.
Natural transduction of normal nuclei ensures orderly replacement of dystrophin and related proteins at the cellular level in DMD. This ideal gene transfer procedure is unique to muscle. After all, only myoblasts can fuse and only muscle fibers are multinucleated in the human body. By harnessing these intrinsic properties, MTT transfers all normal genes to effect genetic repair. Since donor myoblasts also fuse among themselves to form normal fibers in MTT, the muscles benefit from the addition of genetically normal cells as well.
MYOBLAST THERAPY IS THE MEDICINE OF THE FUTURE
Health is the well-being of all body cells. In hereditary or degenerative diseases, sick cells need repairing and dead cells need replacing for health maintenance.
Cell culture is the only way to generate new, live cells that are capable of surviving, developing and functioning in the body after transplantation, replacing degenerated cells that are lost.
Myoblasts are the only cells in the body capable of natural cell fusion. The latter allows the transfer of all of the normal genes into genetically defective cells to effect phenotypic repair through complementation. MTT on DMD is the first human gene therapy demonstrated to be safe and effective. The use of MTT to transfer any other genes and their promoters/enhancers to treat other forms of diseases is underway. Myoblasts are efficient, safe and universal gene transfer vehicles, being endogenous to the body. Since a foreign gene always exerts its effect on a cell, cell therapy will always be the common pathway to health. After all, cels are what life is made of.
DMD: A SAMPLE DISEASE
DMD is a hereditary, degenerative, debilitating, fatal, and undesirable mammalian disease. It is characterized by progressive muscle degeneration and loss of strength. These symptoms begin at 3 years of age or younger and continue throughout the course of the disease. Debilitating and fatal, DMD affects 1 in 3300 live male births, and is the second most common lethal hereditary disease in humans. DMD individuals are typically wheelchair-bound by age 12, and 75% die before age 20. Pneumonia usually is the immediate cause of death, with underlying respiratory muscle degeneration and failure of DMD individuals to inhale sufficient oxygen and to expel lung infections. Cardiomyopathic symptoms develop by mid-adolescence in about 10% of the DMD population. By age 18, all DMD individuals develop cardiomyopathy, but cardiac failure is seldom the primary cause of death.
Before 1950, over 80 chemicals were evaluated and 33 were reported as potentially beneficial (Milhorat, A. T., Medical Annals of the District of Columbia, 23:15 (1954)). None are currently being used (Wood, D. S., In: Kakulas, B. A. and Mastaglia, F. L., eds.: Pathogenesis and therapy of Duchenne and Becker muscular dystrophy. New York: Raven Press; 85-99 (1990)). Unconfirmed therapeutic benefits in DMD have been reported with vitamins, amino acids (Van Meter, J. R., Calif. Med., 79:297 (1953)), ATP (Nakahara, M., Arzneim. Forsch., 15:591(1965)), coenzyme Q (Folkers, K. et al., Excerpta Med. Int. Congr. Ser., 334:158 (1974)), adenylosuccinic acid (Bonsett, C. A., Indiana Medicine, 79:236 (1986)) and growth hormone inhibitor (Coakley, J. H. et al., Lancet, 1(8578): 184 (1988)). Several hundred drugs have been screened (Wood, supra), with some studies showing consistent benefits from steroids (Entrikin, R. K. et al., Muscle Nerve, 7:130-136 (1984)).
The beneficial effects of prednisone on DMD was first reported almost 20 years ago (Drachman, D. B. et al., Lancet, 2:1409-1412 (1974)). The researchers reported that prednisone could delay the degenerative process and in some cases even transiently strengthen DMD muscles. The evidence substantiating prednisone is not without debate (see Munsat, T. L. and Walton, J. N., Lancet, 1:276-277 (1985); Rowland, L. P., Lancet, 1:277 (1975); and Siegel, I. M. et al., I. M. J., 145:32-33 (1974)). Although the mechanism(s) through which prednisone mediates its effect is undefined. Prednisone causes numerous side-effects, and prolonged use induces adverse reactions that by far out-weigh the questionable benefits reported.
Gene manipulation and transfer are other approaches that are being used to treat hereditary and degenerative diseases. However, it will be quite some time before this type of treatment finds clinical application for DMD (Law, P. K., In: Kakulas, B. A. et al., eds.: Pathogenesis and therapy of Duchenne and Becker muscular dystrophy. New York: Raven Press, 190 (1990); and Watson, J. D. et al., Recombinant DNA. New York: W. H. Freeman and Co.; 576 (1992)). Success claimed over intramuscular DNA injections (Acsadi, G. et al., Nature, 352: 815-818 (1991); and Wolff, J. A. et al., Science, 247:1465-1468 (1990)) and arterial delivery of immature muscle cells, also known as myoblasts, to skeletal muscle (Neumeyer, A. M. et al., Neurology, 42:2258-2262 (1992)) is very limited and questionable. Attempt of using transfected autologous myoblasts has resulted in low efficiency and mutation in transfection (Barr, E. and Leiden, J. M., Science, 254: 1507-1509 (1991); Dhawan, J. et al., Science, 254: 1509-1512 (1991); and Smith, B. F. et al., Mol. Cell. Biol., 10: 3268-3271 (1990)). Such approach will yield insufficient myogenic cells to provide for a whole body myoblast transfer therapy (MTT) to treat DMD patients (Law, supra). Clinical trials are currently underway for cystic fibrosis (CF) based on transgenic mice studies (Hyde, S. C. et al., Nature, 362: 250-255-(1993)). Clinical trials with gene therapy have also been attempted on severe combined immunodeficiency. (SCID). Unlike CF and SCID whose genetic defects are mediated through enzymic deficiencies, the genetic defect of DMD manifested as the absence of a structural protein called dystrophin in the cell membrane rather than a regulatory protein.
Although dystrophin serves as a good genetic/biochemical marker (Hoffman, E. P. et al., Cell, 51: 919-928 (1987)) in the evaluation of muscle improvements, dystrophin replacement constitutes only part of the treatment process. This has already been demonstrated, among others, by the present inventor using MTT in mdx mice (Chen, M. et al., Cell Transplantation, 1:17-22 (1992); Karpati, G. et al., Am. J. Pathol., 135: 27-32 (1989); and Patridge, T. A., et al., Nature, 337:176-179 (1989)) and in humans (Gussoni, E., et al., Nature, 356: 435-438 (1992); Huard, J. et al., Clin. Sci., 81:287-288(1991); Huard, J. et al., Muscle Nerve, 15:550-560 (1992); Law, P. K. et al., Lancet, 336:114-115 (1990); Law, P. K. et al., Acta Paediatr. Jpn., 33:206-215 (1991); Law, P. K. et al., Adv. Clin. Neurosci., 2:463-470 (1992); Law, P. K. et al., In: Angelini, C. et al., eds. Muscular dystrophy research. New York: Elsevier Science Publishers, 109-116 (1991); and Law, P. K. et al., Acta Cardiomiologica, 3:281-301 (1991)). Because DMD pathology is one of muscle degeneration and weakness, structural and especially functional improvements are of primary concern. These two parameters have been extensively studied using the dy.sup.2J dy.sup.2J dystrophic mouse as an animal model of hereditary muscle degeneration (Law, P. K., Exp. Neurol., 60:231-243 (1978); Law, P. K., Muscle Nerve, 5:619-627 (1982); Law, P. K. et al., Transplant Proc., 20:1114-1119 (1988); Law, P. K. et al., In: Griggs, R. C.; Karpati, G., eds. Myoblast Transfer Therapy. New: Plenum Press; 75-87 (1990); Law, P. K. et al., Muscle Nerve, 11:525-533 (1988); Law, P. K. et al., In: Kakulas, B. A.; Mastaglia, F. L., eds. Pathogenesis and therapy of Duchenne and Becker muscular dystrophy. New York: Raven. Press; 101-118 (1990); and Law, P. K. and Yap, J. L., Muscle Nerve, 2:356-363 (1979)). These studies lead to the first MTT clinical trial or single muscle treatment (SMT) (Gussoni, E. et al., Nature, 356:435-438 (1992); Huard, J. et al., Clin. Sci., 81:287-288 (1991); Huard, J. et al., Muscle Nerve, 15:550-560 (1992); Law, P. K. et al., Lancet, 336:114-115 (1990); Law, P. K. et al., Acta Paediatr. Jpn., 33:206-215 (1991); Law, P. K. et al., Adv. Clin. Neurosci., 2:463-470 (1992); Law, P. K. et al., In: Angelini, C. et al., eds. Muscular dystrophy research. New York: Elsevier Science Publishers: 109-116 (1991); and Law, P. K. et al., Acta Cardiomiologica 3:281-301 (1991)).
The feasibility, safety, and efficacy of MTT were assessed by this inventor in experimental lower body treatments involving 32 DMD boys aged 6-14 years of age, half of whom were non-ambulatory (Law, P. K. et al., Cell Transplantation, 2;485-505 (1993)). Through 48 injections, 5 billion (55.6.times.10.sup.6 /mL) normal myoblasts were transferred into 22 major muscles in both lower limbs in each of the subjects. Results at 9 months after MTT indicated, interalia, that (1) MTT is a safe treatment; (2) MTT improves muscle function in DMD; and (3) more than 5 billion myoblasts are necessary to strengthen both lower limbs of a DMD boy between 6 and 14 years of age.
OTHER DISEASE CONDITIONS
Potentially every genetic disease can be benefited by MTT. Through natural cell fusion, donor myoblasts insert full complement of normal genes into genetically abnormal cells to effect repair. Promoters and enhancers of the defective gene can be supplied or activated or repressed to achieve gene transcription and translation with the release of hormone(s) or enzyme(s) from transplanted myogenic cells. Likewise, structural protein(s) can be produced to prevent or to alleviate disease conditions.
Alternatively transduced myoblasts can be used. The procedure consists of a) obtaining a muscle biopsy from the patient, b) transfecting a "seed" amount of satellite cells with the normal gene, c) confirming the myogenicity of the transfected cells, d) proliferating the transfected myoblasts to an amount enough to produce beneficial effect and e) administering the myoblasts into the patient.
Retroviral vectors have been used to transfer genes into rat and dog myoblasts in primary cultures under conditions that permit the transfected myoblasts to differentiate into myotubes expressing the transferred genes (Smith, B. F. et al., Mol. Cell Biol., 10:3268-3271 (1990)). Furthermore, mice injected with murine myoblasts that are transfected with human growth hormone (hGH) show significant levels of hGH in both muscle and serum that are stable for 3 months (Dhawan, J. et al., Science, 254:1509-1512 (1991); Barr E. and J. M. Leiden, Science, 254:1507-1509 (1991)).
The transduced myoblast transfer was inspired by a similar approach on adenosine deaminase (ADA) deficiency. In the latter situation, T cells from the patient were transfected with functional ADA genes and returned to the patient after expansion in the number of the transfected cells through cell culture (Culver, K. W. et al., Transpl. Proc., 23:170-171 (1991)).
Similar approach has already been tested in animals using genetically transduced myoblasts to treat hemophilia B (Yao, S. N. et al., Gene Therapy, 1:99-107 (1994)), cardiomyopathy (Marelli, D., Cell Transplantation, 1:383-390 (1992); Koh, G. Y. et al., J. Clin. Inves., 92:1548-1554 (1993)), anemia (Hamamori, Y. et al., Human Gene Therapy, 5:1349-1356 (1994)). Undoubtedly, there will be many hereditary diseases to which myoblast therapy will apply.
Although differentiated, myoblasts are nonetheless embryonic cells that are capable of de-differentiated or even converted. Thus, myoblasts have recently been shown to be converted into osteoblasts with bone morphogenetic protein-2 (Katagiri, T. et al., J. Cell Biol., 127:1755-1766 (1994)). This study demonstrates that cytocline-converted myoblasts can be administered to patients with bone/cartilage degenerative diseases. Alternatively, it has been demonstrated that mouse dermal fibroblasts can be converted to a myogenic lineage (Gibson, A. J. et al, J. Cell Sci., 108:207-214 (1995)).
The implicated usage of myoblast transfer therapy to treat cancer and type II diabetes mellitus is described below.
WHY MYOBLAST THERAPY
In hereditary or degenerative diseases, gene defects cause cells to degenerate and die with time. An effective treatment must not only repair degenerating cells, but replenish dead cells as well. This can best be achieved by the transplantation of genetically normal cells, or somatic cell therapy. The advent of molecular genetics favors single gene manipulation which is currently being explored to treat genetic diseases. Like pharmaceuticals, single gene manipulation cannot replenish lost cells. Further, there is very limited evidence that these approaches can repair degenerating cells.
In U.S. Pat. No. 5,130,141, this inventor disclosed for the first time compositions and methods for treating muscle degeneration and weakness. A composition comprised of genetically normal myoblasts from a donor was injected into one or more of the muscles of a host having a hereditary neuromuscular disorder. Muscle structure and function were greatly improved with the injection, thereby preventing or reducing muscle weakness which is a primary cause of crippling and respiratory failure in hereditary muscular dystrophies. This transplantation of genetically normal muscle cells into the diseased muscles of patients with hereditary muscular dystrophy is known as MTT.
MTT differs significantly from the conventional single gene transfer format in several respects. In this latter gene therapy, single copies of the down-sized dystrophin gene are transduced as viral conjugates into the mature dystrophic myofibers in which many proteins, both structural and regulatory, are lost previously. Multiple gene insertion is necessary to replace these lost proteins (FIG. 1). More gene insertion is needed to produce the cofactors to regulate and to express these lost proteins in order to repair the degenerating cell.